The long term objective of this project is to understand the detailed permeation and gating properties of the inositol 1,4,5- trisphosphate receptor (InsP3R) and the mechanisms and kinetics of its regulation. The InsP3R is a ligand-gated Ca2+ channel expressed in endoplasmic reticulum (ER) membranes in possibly all cell types, releasing stored Ca2+ into the cytoplasm upon binding InsP3, generated in response to numerous extracellular signals. InsP3R-mediated [Ca2+]i signals regulate diverse cell physiological processes, and are precisely controlled in both time and space, being manifested as oscillations and waves, and exhibiting unusual features including quantal release. The InsP3Rs are a family of proteins with diverse expression, suggesting that cells require distinct InsP3Rs to regulate their functions. However, the functional correlates of this diversity are still unknown, primarily because the InsP3R in its normal membrane environment has not been accessible for patch clamping. The major limitation in understanding the mechanisms underlying oscillations, wave propagation, and quantal release has been the lack of precise data on the kinetics of the effects of modulators, including Ca2+ InsP3 and accessory proteins, on the permeation and gating properties of the InsP3R channel. We have overcome these limitations by developing a novel nuclear patch clamp protocol for studying the single channel properties of the endogenous Xenopus InsP3R in its native membrane environment. Our preliminary investigations have revealed new conductance states and voltage-dependent and -independent kinetics, novel regulation, and have suggested the existence of InsP3R clustering and functional interactions among channels. The specific aims of this proposal are to determine the role of the InsP3R in mechanisms underlying quantal Ca2+ release from intracellular stores by characterizing the regulation of the single channel properties of the Xenopus InsP3R by cytoplasmic and lumenal [Ca2+] and by InsP3. We will also determine the ability of the InsP3R to behave as an integrator of cell signals by characterizing the responses of the gating and permeation properties of single InsP3R channels to intracellular regulatory mechanisms, particularly immunophilin binding and calcium- dependent phosphorylation/dephosphorylation. We will use biochemical and molecular expression approaches to examine the interactions between the InsP3R and potential regulatory molecules in the ER lumen and cytoplasm. Finally, we will explore whether InsP3Rs cluster, by analyzing their ultrastructural localization, and determine the functional consequences of clustering for single channel gating and regulation.